Academic journal article Iranian Journal of Public Health

Escherichia Coli and Shigella Genus Detection in Water Sample by Pentaplex Pcr

Academic journal article Iranian Journal of Public Health

Escherichia Coli and Shigella Genus Detection in Water Sample by Pentaplex Pcr

Article excerpt

Background: The aim of this study was to determine the most ubiquitous Escherichia coli specific genes to apply as a genetic marker to speed up the detection time and the sensi- tivity and introduce a multiplex PCR to detect and screen this marker organism in water sample as soon as possible. Since Shigella species share great sequence similarity, so we included a specific gene corresponding to putative integrase enzyme to differentiate Shigella species from Escherichia coli .

Methods: Out of 1956 water samples, one hundred and six Escherichia coli strains, isolated from different water sources of Tehran province (Jalalieh water treatment center, kan water treatment center, number 3, 4 and 5 water treatment centers) within a 1 year were characterized by standard methods based on fast lactose fermentation and IMViC tests. All the isolates were kept in glycerol at -70°C until the final procedure.We extracted DNA from all isolates applying Accuprep Bioneer column kit. New specific sets of primers including structural genes like uspA, phoE and enzyme coding ones such as gadA/B, uidA for Escherichia coli and put int for Shigella were designed in a novel pentaplex PCR by Allele ID 7.6 (Table 1). Following checking the specificity of any set of primers in NCBI, dimer formation and primers interaction, applying Oligo analyzer 1.03, we optimized the multiplex at different temperatures and DNA concentration gradients. …

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